Up to now, recombinant protein expression systems in many hosts such as bacteria, yeasts, insects, animal and plant cells, and transgenic animals and plants and cell-free translation systems have been established. Particularly in production of recombinant proteins by mammalian cultured cells, the proteins are subjected to suitable posttranslational modification, and thus this production system is becoming a standard system for production of therapeutic agents. However, the protein synthesis level in this system is lower than in the system with microorganisms as the host, thus necessitating a larger culture chamber, which would cause shortage of production facilities in biotechnology industry pursuing new medicines (Garber, K., Nat. Biotech. 19, 184-185, 2001). Protein production techniques using transgenic animals and plants attempted to improve production efficiency in recent years still do not attain full confidence (Garber, K., Nat. Biotech. 19, 184-185, 2001).
In the recombinant protein expression systems developed so far, it is often difficult to obtain a large amount of active protein. If a desired protein is toxic to the host to a certain degree, synthesis of the protein is inhibited to decrease the expression level. Further, even if the desired protein is expressed as soluble protein, the protein may be decomposed by proteases in the host so that the amount of the protein produced is reduced to a very low level. In addition, even if the desired protein is expressed, the protein may fail to achieve suitable folding, resulting in formation of an inclusion body. In this case, even if the protein is solubilized and folded again, the amount of the finally obtained active protein is very low. Particularly when a cell-free translation system is used, the inclusion body is easily formed.
When the inclusion body is formed, it is attempted to solve this problem by using a method of expressing the protein in the form of a fusion protein with e.g. glutathione-S-transferase (GST) (Smith, D. B., et al., Gene 67, 31-40, 1988), with thioredoxin (LaVallie, E. R. et al., Bio/Technology 11, 187-193, 1993), or with a maltose-binding protein (Guan, C., et al., Gene 67, 21-30), but there are few cases where formation of the inclusion body is suppressed at high efficiency. Alternatively, there is a method wherein a desired protein is co-expressed with a chaperonin i.e. a protein group supporting protein-folding reaction to increase the amount of the desired protein expressed in the soluble fraction (Nishihara et al., Apply. Environ. Microbiol., 64, 1694-1699, 1998), but at present, this method cannot achieve a remarkable increase in the amount of the active protein.
As a method of solving the problem of decomposition of the desired protein by proteases in the host, a method of using a host deficient in a part of protease structural genes, for example in lon, ompT etc. in the case of E. coli, has been devised (Phillips et al., J. Bacteriol. 159, 283-287, 1984), there are few cases where the influence of decomposition with proteases can be avoided, while if the host is made deficient in all proteases, other problems can occur, thus failing to essentially solve the problem of decomposition with proteases.
As described above, the conventional protein expression techniques have serious problems such as toxicity to hosts, decomposition with host proteases, and formation of inclusion bodies, and thus the expression level is significantly varied depending on the type of protein to be expressed, and expression conditions for each protein should be examined in trial and error. Accordingly, there is demand for development of techniques for essentially solving the problems described above.